Polyglutamine repeat sequences

ABSTRACT

Several neurodegenerative diseases result from the aggregation of polyglutamine repeat proteins into insoluble neuronal intranuclear inclusions. The invention provides methods with which to study the processes of these diseases, including methods for solubilizing polypeptides containing a polyglutamine repeat sequence, for storing these polyglutamine polypeptides and inhibit their spontaneous aggregation, for making the aggregates of polyglutamine polypeptides, for assaying the extension of existing polyglutamine aggregates, for determining the ability of a chemical compound to inhibit aggregation, and for inhibiting aggregation of polyglutamine polypeptides. The invention further provides materials with which to study these diseases including a synthetic aggregate that have a capability to recruit additional monomeric polyglutamine polypeptides and chemical compounds that inhibit the formation and/or extension of polyglutamine aggregates.

This application is a divisional of U.S. patent application Ser. No.10/082,673, filed on Feb. 22, 2002.

FIELD OF THE INVENTION

The invention relates to the field of diseases that are associated withabnormal aggregation of proteins. Specifically, the invention relates tothe field of diseases related to aggregation of intracellular proteinsthat contain polyglutamine repeat sequences.

BACKGROUND OF THE INVENTION

Insoluble aggregates of normally well-behaved proteins are associatedwith a variety of disease states, including the various forms ofamyloidosis, such as Alzheimer's disease, and the prion diseases such asscrapie and bovine spongiform encephalomyelitis, also known as Mad CowDisease. A family of proteins, called molecular chaperones, exists incells to overcome the intrinsic propensity of polypeptides to aggregateduring the normal folding process. However, under certain circumstancesproteins such as the amyloid β (Aβ) peptide associated with Alzheimer'sdisease form insoluble protein aggregates in spite of the presence ofchaperones. In some amyloid diseases, aggregates appear to be toxicsimply by virtue of the effect of their accumulated mass in interferingwith normal tissue function. In neurodegenerative diseases likeAlzheimer's and Huntington's Diseases, the toxic effect appears to bemuch more subtle. According to one hypothesis, aggregates become toxicwhen they clog the cell's normal machinery for clearing aggregates andother superfluous proteins. Another hypothesis holds that aggregatetoxicity derives from the ability of aggregates to recruit otheressential proteins and in the process deplete the normal environment oftheir activities. In any case, it would be of considerable value todevelop ways of removing these aggregates in a benign way, or preventingtheir formation, in analogy to the actions of molecular chaperones.

Analogously to the situation in Alzheimer's Disease and Aβ peptide,there are at least eight inherited neurodegenerative diseases, includingHuntington's disease (HD), spinal and bulbar muscular atrophy (SBMA),dentatorubral pallidoluysian atrophy (DRPLA), and spinocerebellarataxias 1, 2, 3, and 6, that are linked to a particular type of proteinaggregate. Although each of these diseases is associated with adifferent protein, the proteins share the common feature of containingwhat is referred to as an expanded polyglutamine (poly(Gln)) repeat.These poly(Gln) expansion-related diseases, often referred to as CAGrepeat diseases because the glutamine in the poly(Gln) peptide is codedfor by the nucleotides CAG, are progressive disorders characterized bymotor and/or cognitive impairments and distinctive pathological patternsof neuronal degeneration. The only mutation implicated in these diseasesis an expansion of a poly(Gln) sequence in the disease-related protein,generally from a benign length of less than 37 Gln (also referred to asQ₃₇) to a pathological length of Q₃₇ or more.

All of these neurodegenerative disorders present a common feature: theaggregation of the poly(Gln) repeat disease-related protein intoinsoluble neuronal intranuclear inclusions, which has become theneuropathological signature of poly(Gln) disorders. The important rolethat long poly(Gln) repeats play in poly(Gln)-related disorders has beenconfirmed in a number of models in which mutant forms of various diseaseproteins were expressed in transgenic mice, Drosophila, or the nematodeCaenorhabditis elegans.

Although these diseases exhibit similar physiological abnormalities, theonly common features of disease-related proteins are the poly(Gln)domains. Because of this, the expanded poly(Gln) is believed to beresponsible for the pathogenesis. As discussed above, their toxicity isbelieved to be due to their ability to recruit other critical cellularproteins, via their own poly(Gln) components, into the growingaggregate. The loss of protein activity due to this sequestrationappears to be toxic to the cell.

Much about these poly(Gln) diseases remains to be learned. One problemin studying these diseases is that poly(Gln) peptides having a Glnrepeat of more than Q₃ 5 are poorly soluble when transferred directlyinto denaturing solvents or water. In some circumstances, such asdescribed in Sharma, FEBS Letters, 456:181-185 (1999), the insolubilityof long poly(Gln) repeats has presented such an insurmountable problemthat studies had to performed on shorter soluble poly(Gln) repeats suchas Q₂₂, even though mutant proteins involved in the spino-cerebellarataxia type 1 (SCA) are at least Q₄₀.

One method to increase the solubility of monomeric (non-aggregated) Aβpeptide, as disclosed in Evans et al., Proc. Natl. Acad. Sci.,92:763-767 (1995), is to dissolve the peptide in a non-volatiledisaggregating solvent such as dimethyl sulfoxide (DMSO). This methodhas the disadvantage in that the DMSO remains as a permanent co-solventin the final reaction mixture. Therefore, any results obtained instudies of Aβ peptide dissolved in this way may be biased by thepresence of the DMSO.

Use of a volatile disaggregating solvent, trifluoroacetic acid (TFA), tosolubilize the Aβ peptide is disclosed in Jao et al., Amyloid: Int. J.Exp. Clin. Invest., 4:240-252 (1997). Volatile disaggregating solventshave an advantage over non-volatile solvents such as DMSO because theyare readily removed from the peptide, and thus do not interfere withstudies on the peptide. According to this method, TFA is added to thepeptide in a glass container at about a 1:1 ratio (ml TFA:mg peptide).Then the TFA and peptide are sonicated, while adding additional TFA,until the peptide completely dissolves. The TFA is then removed with drynitrogen gas, leaving a coating of peptide on the walls of thecontainer. Trace amounts of the TFA are removed by adding distilledhexafluoroisopropanol (HFIP), sonicating, and removing the HFIP with drynitrogen gas. Sequential TFA-HFIP treatment has also been disclosed inZagorski et al., Meths. Enzymol., 309:189-204 (1999). In this protocol,the role played by HFIP seems to be to simply aid in the removal of TFA,which otherwise will make an aqueous solution of the processed peptideacidic and possibly encourage its reaggregation. In our laboratory, thisprotocol was determined to be effective at solubilizing anddisaggregating peptides in the range of Q₁₅-Q₃₅. However, it is poorlyeffective with peptides greater than Q₃₅.

The inability to dissolve and disaggregate poly(Gln) of the pathologicallength of Q₃₇ or more represents a major obstacle in studying poly(Gln)diseases. A substantial need exists for a method to solubilize and todisaggregate these peptides, and to maintain these peptides in thedisaggregated monomeric state.

Another impediment to the study of poly(Gln) aggregation diseases hasbeen the difficulty in making the aggregates in vitro. Typically, asdisclosed by Scherzinger et al., Cell, 90:549-558 (1997) and Scherzingeret al., Proc. Natl. Acad. Sci., 96:4604-4609 (1999), such aggregates aremade by recombinantly producing a fusion protein (GST-HDex1) containingglutathione S-transferase and exon 1 of the HD (Huntington's Disease).When the fusion protein is cleaved with trypsin, a high molecular weightprotein aggregate with a fibrillar or ribbon-like morphology similar tothose found in scrapie and in Alzheimer's disease are formed. Theserecombinantly produced aggregates do not completely correlate with thenatural poly(Gln) disease state.

In the disease state, peptides with poly(Gln) repeat lengths as low asQ₁₅ or Q₂₀, while of insufficient length to readily spontaneouslyaggregate, readily add to pre-existing aggregates. As disclosed inPerutz, et al., Proc. Natl. Acad. Sci. USA, 91:5355-5358 (1994),aggregates having such shorter poly(Gln) peptides differ from those seenin Alzheimer's disease. Polyglutamine aggregates made in vitro can adopta number of morphological forms, each of which may differ in toxicactivity. Very little is known for certain about the morphology of thetoxic form of polyglutamine aggregates in vivo, but if the recruitmenthypothesis is correct, than the aggregates must be especially potent inthis activity. Consequently, a substantial need exists for methods toprepare different kinds of poly(Gln) aggregates in vitro in order toidentify those that are particularly active in supporting depositionand/or extension of additional polyglutamine peptides.

Given the potential role of poly(Gln) aggregates and poly(Gln) aggregateextension in the pathogenesis of expanded CAG repeat diseases, it isessential to characterize the fundamental aggregation behavior ofpoly(Gln) sequences. Studies of the aggregation behavior dependence onpoly(Gln) repeat length are important to fully understand thecorrelation between length and disease risk, as well as the rules thatcontrol the recruitment of other poly(Gln)-containing peptides andproteins into growing poly(Gln) aggregates. Presently, no assay existsthat allows the observation of both the homologous growth of anaggregate as well as the ability of the aggregate to recruit otherpolyglutarnine peptides into the aggregate via its extension. Such anassay would have use as an assay for poly(Gln) aggregation andrecruitment and as a screen for aggregation inhibitors as potentialtherapeutics. The assay would also be capable of detecting“extension-competent” or “seeding-competent” aggregates in tissue andserum samples that might be crucial for diagnosis and for evaluating therole of poly(Gln) aggregates in the disease mechanism.

SUMMARY OF THE INVENTION

In one embodiment, the invention is a method for solubilizing and/ordisaggregating a polypeptide. The method of the invention is especiallyuseful for solubilizing and/or disaggregating a peptide that has atendency to spontaneously aggregate. Such peptides, and aggregatesthereof, are found in certain diseases such as prion disease andAlzheimer's disease, and with CAG repeat (poly(Gln)) diseases.

According to this embodiment of the invention, the peptide is combinedwith a mixture of trifluoroacetic acid (TFA) and hexafluoroisopropanol(HFIP) and the peptide is permitted to dissolve in the mixture.Utilizing a mixture of these two solvents, rather than alone orsequentially as taught in the prior art, has been unexpectedlydiscovered to increase the solubility of peptides, even those havinglong repeating sequences, such as poly(Gln) where Q>35, that areotherwise difficult or impossible to dissolve by presently availablemethods. This permits the subsequent step of removing the TFA and HFIPand then resolubilizing the monomeric peptides in water so that thepeptides will be in a useful form. According to this embodiment of theinvention, it is preferred but not essential that the poly(Gln)polypeptide to be solubilized contain charged amino acids flanking thepoly(Gln) sequence in order to increase its kinetic solubility inaqueous solution, that is the transient solubility of the polypeptidewhen initially dissolved.

In another embodiment, the invention is a method for storing monomericpeptides that have a tendency to aggregate. According to this method,solubilized disaggregated peptides are snap frozen at a low temperaturewhere freezing occurs essentially instantaneously and stored at atemperature of below −50° C., preferably about −80° C. or lower. Ifdesired, DMSO may be added to the solution containing the monomericpeptides prior to freezing. The presence of DMSO, although notpreferred, may further help to prevent aggregation of the peptides.

In another embodiment, the invention is a method for making an aggregateof peptides that are prone to aggregation, such as peptides containing aglutamine repeat sequence. According to this embodiment, anaggregation-prone peptide, such as a polypeptide having a glutaminerepeat sequence, which peptide is in solution, is frozen and incubatedin the frozen state at a temperature at which aggregate formation mayoccur, and then thawed, at which point the aggregates are collected. Themethod of the invention permits the formation of peptide aggregates thatare not otherwise obtainable, or that are impractical to produce, byprior art methods, such as poly(Gln) aggregates of Q₁₅. If desired, theaggregates thus formed may be further processed by sonication and/orfiltration.

In another embodiment, the invention is an vitro-produced aggregatecomposed of peptides having a glutamine repeat sequence, wherein theaggregate is in the shape of a filament having a diameter of less than10 nm and a length of less than 100 nm. The aggregate may be produced bythe method of the invention for making an aggregate of peptidescontaining a glutamine repeat sequence. Preferably, there is amultiplicity of aggregates of which the majority of the aggregates inthe multiplicity of aggregates are in the shape of a filament having adiameter less than 10 nm and a length of less than 100 nm.

In another embodiment, the invention is an in vitro assay for poly(Gln)aggregate formation and for the extension of existing poly(Gln)aggregates. Present methods of assaying for poly(Gln) aggregateformation depend on measurements of the total mass of an aggregate, andare therefore limited in their ability to provide detailedquantification of heterologous aggregation reactions. The assay of theinvention is capable of monitoring the repeat length dependence ofheterologous aggregation extension, that is, the ability of a poly(Gln)peptide to assemble into an aggregate of another poly(Gln) peptide.

According to this embodiment, labeled monomeric (non-aggregated)poly(Gln) peptide in solution is added to a support to which are fixedpoly(Gln) aggregates so that the monomeric peptide solution contacts thefixed aggregates, and the amount of labeled monomeric peptide that bindsto the aggregate is determined, typically by counting the amount of thelabeling that remains following removal of any unbound monomericpeptide. Preferably, following the addition of the monomeric peptide tothe container, the aggregates and monomeric peptides are incubated for apredetermined amount of time during which time the aggregates andmonomeric peptides have an opportunity to bind to one another. Alsopreferably, at a time following the addition of the monomeric peptideand before the amount of labeling remaining is determined, the containeris rinsed to remove any monomeric peptide that does not bind to theaggregates. Preferably, the poly(Gln) aggregates that are fixed to theinner surface of the container are the poly(Gln) aggregates of theinvention.

In another embodiment, the invention is a method for determining theability of a chemical compound to inhibit the expansion of existingpoly(Gln) aggregates by either homologous or heterologous extensionreactions. According to this embodiment, the assay of the invention isperformed wherein a test compound is exposed to either or both of thefixed poly(Gln) aggregates and the monomeric poly(Gln) peptides. In thismanner, the ability of the test compound to inhibit the formation of, orthe extension of, poly(Gln) aggregates can be determined.

In another embodiment, the method of the invention is a method forinhibiting the formation of, or expansion of, poly(Gln) aggregates.According to this embodiment, a poly(Gln) aggregate is exposed to achemical compound that has the capability to inhibit the formation of,or the expansion of, poly(Gln) aggregates, which capability ispreferably determined by testing according to the assay of theinvention, thereby inhibiting the expansion of the poly(Gln) aggregate.Preferably, the chemical compound is a compound, or combination ofcompounds, that has been determined by the assay of the invention tohave the capability to inhibit the formation of, or the expansion of,poly(Gln) aggregates. In the previous sentence, the term “compound”includes homologs and analogs of compounds that have been determined tohave the capability to inhibit the formation of, or the expansion of,poly(Gln) aggregates, and which homologs and analogs also have thiscapability. It is conceived that this method of the invention may beuseful in vitro, for example as a research tool to study the formationor extension of polyglutamine aggregates, and in vivo, such as forslowing or preventing aggregation in patients susceptible to orsuffering from a poly(Gln) repeat disease, such as Huntington's Disease.For in vivo use, an effective amount of a chemical compound, orcombination of chemical compounds, that inhibits the formation orextension of poly(Gln) repeats is administered to a patient in needthereof.

In this specification, when referring to a sample containing a poly(Gln)polypeptide of Q_(N), “Q_(N)” indicates that the sample containspoly(Gln) polypeptide having a weighted average of N 5 poly(Gln)repeats, where N is an integer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an electron microscopic image of prior art polyglutamineaggregates.

FIG. 2 shows an electron microscopic image of polyglutamine aggregatesof the invention.

FIG. 3 is a line graph showing the dependence of the rate of the fastphase on biotinyl-poly(Gln) concentration.

FIG. 4 is a pair of line graphs showing the correlation of the signalstrength detected with the amount of poly(Gln) aggregate deposited.

FIG. 5 is a graph showing the correlation between results of the assayof the invention utilizing synthetic poly(Gln) aggregates (circles) andataxin-3(Q₂₇) aggregates (squares) found in polyglutamine repeatdisease.

FIG. 6 is a graph showing the relationship of length of poly(Gln)peptides and the capability of the peptides to bind to existingpoly(Gln) aggregates.

FIG. 7 is a graph showing the effectiveness of aggregates ofpathological size (Q₃₇) to serve as templates for heterologous extensionby other poly(Gln) peptides.

FIG. 8 is a graph showing the solution phase growth of poly(Gln)aggregates.

DETAILED DESCRIPTION OF THE INVENTION

The method of the invention to dissolve and/or disaggregate poly(Gln)peptides overcomes the disadvantage of prior art methods as it permitsthe dissolution and/or disaggregation of poly(Gln) peptides having arepeat length higher than Q₃₅, especially those peptides having apoly(Gln) repeat sequence in the pathological range of Q₃₇ or higher. Inthis specification, when referring to the method and to dissolving anddisaggregating, the term “dissolving” refers to dissolving monomeric oraggregated poly(Gln) peptides and the term “disaggregating” refers todisaggregating aggregated poly(Gln) peptides to produce monomericpoly(Gln) peptides. The term “solubilize” as used herein refers toeither or both dissolving and disaggregating. The method is suitable tosolubilize poly(Gln) peptides having a repeat length of up to Q₄₄ andeven up to Q₅₁ or higher, such as up to Q₆₅ or higher.

This method is suitable for dissolving any peptide, and conceivably fordisaggregating any aggregate, not just those peptides having a poly(Gln)repeat sequence. For example, the method may be used to solubilize anyaggregation-prone peptide, such as the amyloid β peptide or a peptideassociated with a prion disease. An aggregation-prone peptide, forpurposes of this patent, is a peptide which, following dissolution andduring storage in solution at room temperature, spontaneously formsaggregates in solution, which aggregate formation may be determined bydetecting an increase in light scattering through the solution withinone hour from the beginning of the room-temperature storage. Forsolubilizing polypeptides having a poly(Gln) repeat sequence longer thanQ₃₅, it is preferable that the polypeptide contain charged amino acids,such as lysine, on both sides of the poly(Gln) repeat sequence. This hasbeen found to increase the kinetic solubility of the poly(Gln) peptide.

According to this method, rather than using either TFA or HFIP, or asequential combination of TFA and HFIP, to solubilize a peptide, amixture of TFA and HFIP is used. It has been surprisingly discoveredthat even hard to dissolve peptides, such as a peptide having apoly(Gln) repeat sequence higher than Q₃₅, and such as peptides that areincompletely soluble in TFA or HFIP separately, are solubilized by themethod of the invention.

In a preferred embodiment, the peptide is solubilized in a mixture ofTFA and HFIP in a ratio of about 1:1. The ratio of TFA and HFIP does nothave to be precise. Any ratio of TFA and HFIP is suitable so long as thetwo compounds are combined during the peptide-solubilization. Suitableratios of TFA and HFIP are between 20:1 and 1:20, preferably between10:1 and 1:10, and most preferably between 5:1 and 1:5, such as 3:1 to1:3 or 2:1 or 1:2. The most preferred ratio is about 1:1 and bestresults are expected to be obtained when this ratio is used.

Preferably, the peptide is combined with the TFA/HFIP mixture in acontainer to provide an initial concentration of about 0.5 mg of peptideper ml of the mixture. However, the initial concentration is immaterialas any concentration of peptide in the mixture is suitable. Atconcentrations lower than 0.5 mg/ml, typically all of the peptide willsolubilize without adding additional TFA and/or HFIP. At a concentrationof 0.5 mg/ml or higher, typically additional TFA and/or HFIP may have tobe added to completely solubilize the peptide.

Preferably, the peptide/TFA/HFIP mixture is incubated until visualinspection or other determination indicates that the peptide hassolubilized. If desired, the mixture may be vortexed or otherwiseagitated to speed the solubilization process. Incubation is preferablyat room temperature, although the temperature may be higher or lower ifdesired, with the understanding that solutes tend to dissolve morerapidly at higher temperatures than at lower temperatures.

An additional optional step is to remove the TFA and the HFIP from thepeptide. This may be accomplished by any known method to remove volatilesolvents from a solution. A preferred method is to remove the solventsunder a stream of an inert gas, such as nitrogen or argon to leave aresidue of dried peptide on the surface of the container. In so doing,it is preferred to utilize a flow rate of the inert gas that is fastenough to maximize the surface area over which the dried peptide residueis deposited but is not so fast as to lead to splashing of the peptidesolution out of the container.

Following removal of the TFA and HFIP, the disaggregated peptides may beresolubilized. Resolubilization is preferably in water, preferablydistilled, or in a mixture of water and TFA, or in any other medium inwhich the monomeric peptide will dissolve. A preferred pH range forresolubilization of peptides containing a poly(Gln) repeat sequence isabout 3 to 5, and preferably about 3, as these peptides tend toreaggregate more rapidly as the pH approaches 7 or higher. However, pHhigher than 5 may be used for resolubilization if desired.

To verify that all aggregates have been effectively dissolved, thesolubilized or resolubilized peptides, preferably from an aliquot of thepeptide/TFA/HFIP solution, may be tested with a fluorometer and shouldexhibit low 90° light scattering comparable to that of water alone or toa solution of monomeric poly(Gln) in phosphate buffered saline (PBS).When testing for complete dissolution of peptides having poly(Gln)repeat sequences, it is preferred that the fluorometer is set toexcitation and emission wavelengths of about 450 nm. If scattering isfound to be high compared to buffer alone, incubation of the remainingpeptide/TFA/HFIP solution should be continued until testing of furtheraliquots reveals that light scattering has been reduced to backgroundlevels. Alternatively, the peptide/TFA/HFIP mixture can be furtherincubated for several hours, such as up to 4 hours or more, withoccasional swirling or other agitation, until complete solubilization isobtained.

Following resolubilization, if any residual aggregates remain or torigorously ensure that all aggregates have been eliminated, they may beremoved, such as by filtration or centrifugation with retention of thesupernatant. Additionally, if desired to rigorously ensure that allaggregates are eliminated, only the top portion, for example theuppermost ⅔, of the supernatant may be retained.

The method of the invention for solubilizing peptides has beenunexpectedly discovered to have the additional advantage of stabilizingmonomeric peptides in solution, that is in inhibiting the aggregation ofdissolved monomeric peptides. For example, it has been found that a Q₁₅peptide that has been solubilized directly into a pH 3 aqueous bufferand maintained in this buffer tends to rapidly aggregate when the pH isadjusted to 7.4. However, if the peptide is first treated with theTFA/HFIP combination before solubilizing in the pH3 aqueous buffer, thenupon adjusting the pH to 7.4 the peptides will remain in the monomericstate for at least a month or more, even if stored at 37° C. Althoughsuch dramatic results may not be attainable with peptides havingpoly(Gln) repeat sequences longer than Q₁₅ which have an increasetendency to aggregate, it is conceived that solutions of these peptideswill be more stable if solubilized according to the method of theinvention than if solubilized by prior art methods.

Peptides that have a tendency to form aggregates, such aspoly(Gln)-containing peptides, generally exhibit a time-dependentaggregation when stored in suspension or solution. This occurs even atlow pH such as pH 3 and even at low temperatures below physiologic orroom temperature. It has been surprisingly discovered that poly(Gln)aggregates will form during storage in the frozen state, such as between−5° C. or −10° C. and −50° C., and that storage in the frozen state mayactually stimulate aggregation. It has been further surprisinglydiscovered that, according to the method of the invention for storingdisaggregated peptides, snap freezing of solubilized peptides, such aswith liquid nitrogen, followed by storage at a temperature below 50° C.,preferably about −80° C., will inhibit or prevent aggregation and willpreserve the disaggregated state of aggregation-prone peptides, such aspoly(Gln) peptides, for several months.

According to the method of the invention for making an aggregate,dissolved monomeric aggregation-prone polypeptides, such as dissolvedmonomeric peptides having a poly(Gln) repeat sequence of at least Q₁₅ toQ₂₀, preferably of at least Q₃₅, and most preferably of at least Q₃₇,are frozen, such as at between −10° C. to −50° C. or by snap freezing,such as with liquid nitrogen, dry ice, or dry ice-ethanol. The monomericpeptides may be a homogenous or heterogenous population. For example,the poly(Gln) monomeric peptides may be a homogenous population ofpeptides having the same poly(Gln) length, such as all Q₁₅, Q₂₀, Q₃₅, orQ₃₇ or higher, or may be a heterologous population of peptides havingdifferent poly(Gln) lengths, such as a combination of poly(Gln) peptidesof Q₁₅ and higher. In a preferred embodiment, the monomericaggregation-prone peptides used to make the aggregates of the inventionare poly(Gln) peptides that preferably contain a poly(Gln) sequence ofgreater than Q₁₅.

Optionally, the disaggregated peptides may be centrifuged to eliminateany aggregate seeds before freezing. Preferably, but not necessarily,the frozen peptides are stored in the frozen state, preferably at atemperature between −5° C. to −50° C., for at least a day. Preferably,before freezing, the monomeric peptides had been dissolved by the methodof the invention for dissolving peptides.

Following the freezing step, optionally followed by the storing step,the frozen peptides are thawed. Aggregates spontaneously form duringincubation while frozen, and to some extent during the freezing processunless snap frozen.

Aggregates formed by this method differ in structure from aggregatesformed by prior art methods, such as those that do not include afreezing step in accordance with the method of the invention for makingpolypeptide aggregates.

FIG. 1 and FIG. 2 show an electron microscopic comparison ofpolyglutamine aggregates formed without a freezing step (FIG. 1) andformed with the freezing step (FIG. 2). The aggregates, as shown inFIGS. 1 and 2 were fixed to mica grids and negatively stained with a0.25% potassium phosphotungstate solution. The polyglutamine aggregatesmade with the freezing step are in the form of filaments between 1 and10 nm in diameter, typically between 2 and 7 nm, and preferably between2 and 3 nm in diameter. Thus, the polyglutamine aggregates of theinvention are between 2% and 20% of the typical 50 nm width ofpolyglutamine aggregates made without the freezing step. Thepolyglutamine aggregates according to the invention have a lengthgenerally between 20 nm and 100 nm, typically between 30 nm and 75 nm,and preferably between 40 nm and 60 nm, such as 50 nm. Thus, thepolyglutamine aggregates of the invention are about 2% to 20% of thetypical 500 nm or greater length of polyglutamine aggregates madewithout a freezing step. The polyglutamine aggregates of the inventionmade with the freezing step appear to be similar in form to thefilamentous components of the aggregates formed without the freezingstep. It is conceived that the freezing step prevents the furtheraggregation of polyglutamine filaments into a longer and thickerconformation.

Aggregates of the invention may be, if desired, further processed toproduce even finer aggregates. In a preferred method, aggregates of theinvention, as described above, are sonicated, such as with a probesonicator at two minutes at 0° C. The processed aggregates may then befiltered through a membrane, such as between 0.22 μm to 1.2 μm membranefilters, or through a series of membranes, such as sequential 1.2 μm,0.45 μm, and 0.22 μm filters, to obtain a uniform desired aggregatesize. As shown in FIG. 7, aggregates that are sonicated and/or filteredhave an increased capability to act as templates for extension byadditional poly(Gln) peptides.

The aggregates of the invention, either with or without furtherprocessing, are advantageous over prior art aggregates as they areconceived to more closely resemble the polyglutamine aggregates thatform in the naturally occurring disease. Additionally, as shown in FIG.7 and discussed in more detail in the Examples below, the aggregates ofthe invention, when deposited onto a glass or plastic surface such as amicrotiter well, are about 30 times more potent in supporting depositionand/or extension of additional polyglutarnine peptides than are thebroad ribbon polyglutamine aggregates formed without a freezing step andshown in FIG. 1. This property is believed to be due to an increase insurface-exposed growth points in the aggregates of the inventioncompared to the thicker and longer prior art aggregates or possibly dueto a deleterious change in the local structure of growth points forfurther aggregation when the filaments are incorporated into the broadaggregates of the prior art.

According to the assay of the invention, poly(Gln) aggregates areimmobilized on a surface, such as a microtiter well or a glass orplastic slide, and are then incubated with a labeled monomericpolyglutamine peptide. The degree of adherence, that is addition ofmonomeric polyglutamine peptides to the bound poly(Gln) aggregates isdetermined by detecting the amount of label that remains after removalof any unbound monomeric peptide.

The assay of the invention is illustrated below using Q₂₈ and Q₃₇aggregates. However, the actual poly(Gln) aggregate that is used isimmaterial. For example, the aggregates may have a poly(Gln) repeat fromQ₁₅ to Q₅₁, or may be even longer if desired, for example, up to Q₁₀₀.Aggregate extension by this assay can be observed over a wide variety ofpoly(Gln) lengths—although some applications may require peptides of acertain length range for optimal performance in a particularapplication. Additionally, the aggregates may be produced from acombination of poly(Gln) repeat polypeptides, such as Q₁₅, Q₂₀, Q₂₅,Q₂₈, Q₃₅, Q₃₇, or any other combination. Moreover, the aggregates may beformed of polypeptides in which the major portion of the polypeptide isnot a CAG repeat, as is illustrated below with ataxin-3(Q₂₇).

Preferably, the poly(Gln) aggregates that are fixed to the surface arethe aggregates of the invention, or the aggregates made by the method ofthe invention. These aggregates have been determined to be moreefficient at supporting extension reactions, that is recruitment ofadditional polyglutamine peptides into an existing aggregate, than thebroad ribbon-like aggregates of the prior art. However, if desired,prior art aggregates may be used in the assay of the invention.

Monomeric (nonaggregated) polyglutamine peptides are labeled, such aswith a radiolabel or with a non-radioactive label such as biotin, and asolution of the monomeric labeled polyglutamine peptides are incubatedin contact with the immobilized aggregates. The presence and degree ofrecruitment of the monomeric peptides onto the immobilized aggregatesmay be determined by counting the number of labeled peptides that bindto the aggregates, preferably following rinsing to remove any unboundaggregates. From this determination, and with or without knowledge ofthe concentration of monomeric poly(Gln) peptides that are in thesolution, the rate (moles/time) of incorporation of poly(Gln) peptidesinto poly(Gln) aggregates may be calculated.

The assay of the invention may be used, as described above for studyingkinetics of aggregate formation or for discovering inhibitors ofaggregate formation, for example. Another use of the assay is forassaying test samples for the presence of aggregates. The assay of theinvention is extremely sensitive and is capable of detecting very smallquantities of poly(Gln) aggregates by detecting the ability of existingaggregates to recruit additional poly(Gln) peptides. The assay exhibitsexcellent linearity of response (r²=99), with a sensitivity sufficientto detect as little as 40 pg of a synthetic aggregate. This type ofassay may be done, for example, by pouring microplates with differentamounts of aggregate per well and testing how well the amount in thewells serves as a matrix for aggregate extension by monitoring theincorporation of subsequently labeled monomer.

This assay may also be used to analyze a tissue extract samplecontaining an unknown quantity of aggregate. The presence of aggregatein the sample, and possibly the quantification of the amount ofaggregate present, may be determined by incubating a dilution of theextract in the microplate well, then assaying with a standard amount oflabeled monomer, and reading the signal obtained against a standardcurve obtained with synthetic poly(Gln) aggregates, such as those of theinvention. In this way, the assay may be used as a diagnostic tool todetermine the disease status of a patient, either to follow the progressof a poly(Gln) disease in the patient or to monitor the response of thepatient to a therapy.

The assay permits the study of the assembly mechanisms of poly(Gln)aggregates and of critical features of this reaction, such as poly(Gln)length dependence. Another important use of the assay is as a valuabletool in screening and characterizing possible anti-aggregationinhibitors, which inhibitors may be useful therapeutically, and forstudying structure-function relationships of the poly(Gln) aggregationreaction, and inhibitors of the reaction, in a clean, well-definedsystem. In this use of the assay, a known amount of aggregate is boundto a plate, then a known concentration of labeled monomeric poly(Gln)peptide is incubated with the bound aggregate. The assay in this form iscapable of detecting femtomole amounts of monomer that become insolubleby binding to the aggregate.

A number of compounds have been detected, by the assay of the invention,to be useful in inhibiting polyglutamine aggregation and/or recruitmentof additional peptides into existing polyglutamine aggregates. Compoundsthat have been determined to be useful in inhibiting such aggregationinclude polyhydroxy-aromatic compounds such as 6-fluoronorepinephrine,3-(3,4-dihydroxyphenylserine), α-methylnorepinephrine, benserazide,2,10,11-trihydroxyaporphine, and 2,10,11-trihydroxy-N-propylnoraporphine, and2,11-dihydroxy-10-methoxyaporphine. A preferred compound for inhibitingpolyglutamine aggregation is myricetin(3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-1-benzopyran-4-one),disclosed in Merck Index, 9th Ed. entry 6158, pages 821-822 (1976).These and other compounds determined to be useful in inhibiting suchaggregation are useful as in vivo pharmaceutical compounds for thetreatment of expanded CAG repeat diseases. That is, these compounds maybe administered to a patient in need thereof to prevent the extension ofexisting aggregates in the patient and/or to prevent the formation ofaggregates in a patient who is susceptible to forming these aggregates.

The illustrated compounds that inhibit poly(Gln) aggregation shareseveral properties. Each is an aromatic compound containing one or morebenzene rings. Each has more than one hydroxyl groups, with at least oneof the hydroxyl groups being an aryl hydroxyl. Further several, but notall of these inhibitor compounds, have an amino group that is located atsome distance from the at least one of the aryl hydroxyl groups, that isthe amino group is not bound directly to the phenyl group to which thearyl hydroxyl group is bound.

The invention and its various embodiments are further illustrated by thefollowing non-limiting examples.

EXAMPLE 1 Solubilization of poly(Gln) Peptides

Peptides were obtained as unpurified peptides made by custom solid-phasesynthesis from the Keck Biotechnology Center of Yale University. Thesynthetic peptides had a common sequence of a poly(Gln) flanked by pairsof lysine residues (K₂Q_(n)K₂) which bestow a net charge on the peptideat pH 7 to improve initial solubility. Additional biotinylated-K₂Q_(n)K₂peptides were obtained from the Keck Biotechnology Center for the assaydescribed below in the Examples 8 to 10. The biotinylated peptides weremade by N-terminal derivatization during the solid-phase synthesis ofthe peptides.

1 to 5 mg of peptide as a lyophilized powder from the solid-phasesynthesis was combined with a 1:1 mixture of TFA (Pierce, Rockford,Ill.) and HFIP (Sigma, St. Louis, Mo.) to generate a 0.5 mg/mlsuspension in a 20 ml Erlenmeyer flask. The suspension was incubated atroom temperature with vortexing until visual inspection indicated thatthe aggregates had dissolved. The suspension/solution was then furtherincubated for a total time of 0.5 to 4 hours, depending on initialconcentration of peptide. The TFA/HFIP solvent was removed at roomtemperature under a stream of argon gas directed through a Pasteurpipette into the flask. Gas flow was continued for 15 to 30 minutesafter visible removal of the solvent. Immediately after gas flow wasterminated, distilled water adjusted to pH 3 with TFA was added to theflask to provide a peptide concentration of 100 to 200 μM.

EXAMPLE 2 Testing for Complete Solubilization

Aggregates may persist in the TFA/HFIP mixture after all visible tracesof insoluble peptide have disappeared. To check for this, after visuallyapparent solubilization had been achieved, a test aliquot of thepeptide/TFA/HFIP solution was dried under argon and resolubilized asdescribed in Example 1. The dissolution of the aggregates was verifiedwith a fluorometer set to excitation and emission wavelengths of 450 nm.The aqueous 100-200 μM solution exhibited low 90° light scatteringcomparable to water alone.

If scattering is high, incubation of the main solubilization reaction inthe TFA/HFIP is continued, with or without agitation or swirling, untillight scattering when measured by fluorometry is reduced to thebackground level of aqueous buffer alone. If any residual aggregatespersist after the peptide has been dissolved in water, such as at pH ofabout 3, these may be removed by centrifugation and decanting the majorportion of the supernatant. Even if no aggregates are apparent, suchcentrifugation may be advisable to ensure that no aggregates arepresent.

EXAMPLE 3 No Changes in Peptides due to TFA/HFIP

To assess whether prolonged exposure of poly(Gln) peptides to theTFA/HFIP solvent combination might introduce any chemical changes in thepeptide, samples of the peptide K₂Q₁₅K₂ (Q₁₅ flanked by two lysines ateach end) were incubated overnight at room temperature in a 50:50mixture of TFA and HFIP and in TFA and HFIP separately. In excellentagreement with the calculated molecular weight of 2453.68, thereconstructed +1 state parent ion of the starting peptide fromelectrospray mass spectrometry was calculated to be 2352.50. Thecorresponding reconstructed +1 states after incubating in the threesolvent treatments were 2352.40, 2352,40, and 2352.40, respectively. Nonew fragments were observed in the mass spectra after these solventtreatments.

EXAMPLE 4 Spontaneous Aggregation

Poly(Gln) peptide aggregates made of poly(Gln) polypeptides of variouslengths were disaggregated and solubilized as described in Example 1 andthen incubated at a concentration of 57 μg/ml in PBS at 37° C.Aggregation was monitored by light scattering. The results, as shown inFIG. 8, show that the rate of spontaneous aggregation in solution isrelated to the length of the poly(Gln) polypeptides.

Q₁₅ peptides displayed little tendency to aggregate, exhibiting a lagtime of more than 250 hours. Peptides of Q₂₅ to Q₃₂ have lag times inthe range of 25 to 100 hours and the reactions proceed very slowly onceaggregation is initiated. As poly(Gln) repeat length increased into therange of Q₃₇ to Q₄₁, the lag times dropped to about 20 hours and theaggregation reactions proceeded much more rapidly than for the shorterpoly(Gln) peptides. Poly(Gln) peptides of Q₄₄ showed a lag time of onlya few hours and very rapid aggregation reaction following the initialformation of aggregate.

This study was repeated using thioflavin T as described below to monitorthe formation of aggregates. The results were essentially identical. Thestudy was repeated with a concentration of 10 μM of the various lengthsof poly(Gln) aggregates. Results with identical molar concentrationswere similar to results obtained with identical w/v concentrations.

EXAMPLE 5 Prolonged Storage of Solubilized Peptides

Even at pH 3, solubilized peptides exhibit a time-dependent aggregation.To prevent this, following resolubilization, the peptide solution wassnap frozen in liquid nitrogen and was stored at −80° C. It wasdetermined that storage at this temperature preserves the disaggregatedstate of dilute poly(Gln) solutions for several months of storage.Storage at temperatures of about −50° C. or below was found to protectagainst aggregation. Storage at a temperature of −20° was found to beinsufficient to protect against aggregation.

EXAMPLE 6 Preparing poly(Gln) Aggregates Without a Freezing Step

Q₃₇ peptides were solubilized as described in Example 1. The solubilizedpeptides were adjusted to a concentration of 57 μg/ml in PBS andincubated at 37° C. for 7 days. Following this, the concentration ofpeptide was increased to 10 μM and the aggregates were permitted to growat 37° C. Aggregation was monitored by thioflavin T fluorescence, asdescribed in Example 7. Aggregates were collected by centrifugation at20,800×G at 4° C., then resuspended to 44 μg/ml in extension buffer (PBS1×, 0.01% TWEEN-20 and 0.05% NaN₃). The aggregates were fixed to micagrids and negatively stained with a 0.25% potassium phosphotungstatesolution, and analyzed by scanning electron microscopy on a HitachiH-600 electron microscope. See FIG. 1.

EXAMPLE 7 Preparing poly(Gln) Aggregates of the Invention

Q₃₇ peptides were solubilized as described in Example 1. The solubilizedpeptides were adjusted to a concentration of 57 μg/ml in PBS andincubated at 37° C. for 7 days. Following this, the concentration ofpeptide was increased to 10 μM and was incubated at 37° C. for 24 hours.The aggregates were then snap frozen, followed by further incubation at−20° C. for 24 hours, during which incubation poly(Gln) aggregatesspontaneously formed. Aggregation was confirmed by thioflavin Tfluorescence, as described in LeVine, “Quantitation of β-sheet amyloidfibril structures with thioflavin T”, in Amyloid, Prions and otherProtein Aggregates, vol. 309, pages 274-284, R. Wetzel, ed., AcademicPress, San Diego, Calif. (1999). Thioflavin T was then added to thesolution at a concentration of 50 μM, and the fluorescence wasdetermined by excitation at 450 nm (5 nm slit) and emission at 482 nm(10 nm slit). Aggregates were collected by centrifugation at 20,800×G at4° C., then resuspended to 44 μg/ml in extension buffer (PBS 1×, 0.01%TWEEN-20 and 0.05% NaN₃). The aggregates were fixed to mica grids andnegatively stained with a 0.25% potassium phosphotungstate solution, andanalyzed by scanning electron microscopy on a Hitachi H-600 electronmicroscope. See FIG. 2.

EXAMPLE 8 Further Processing of Aggregates of the Invention

Some of the aggregates formed by the processes described in Examples 5and 6 were further processed by sonication with a probe sonicator for 2minutes at 0° C. or by sonication with a probe sonicator for 2 minutesat 0° C. followed by filtration through a 1.2 μm membrane filter. Thefiltrate containing small aggregates was retained.

EXAMPLE 9 Preparation of Aggregate Plates

Poly(Gln) aggregates were fixed to activated ELISA microtiter plates(EIA/RIA Plates, Costar, Atlanta, Ga.) by passive adsorption. Themicroplate was incubated uncovered for 17 hrs at 37° C. with 20 ng ofK₂Q₂₈K₂ aggregate diluted in 100 μl extension buffer. During this timeperiod the wells were permitted to dry out. After the 17 hrs incubation,the wells were washed 3 times with extension buffer, blocked for 1 hr at37° C. with 0.3% gelatin in extension buffer, and washed again 3 times.Following this treatment, the plate was used immediately although it canbe stored for 1 week at 4° C. with 200 μl of extension buffer in thewells.

EXAMPLE 10 Preparation of Biotinylated poly(Gln) Peptides

Biotinyl-K₂Q₂₈K₂ was solubilized as described above in Example 1. Theconcentration of the H₂O/TFA pH3 biotinyl-K₂Q₂₈K₂ solution wasdetermined by comparing to a concentration standard that was establishedusing a K₂Q₁₅K₂ solution, whose concentration was determinedindependently by amino acid composition analysis. The calibratedsolution was saved as a standard for HPLC determination of futurepeptide concentrations.

A known volume of the standard was injected on an HPLC and its elutionat A215 to A220, the region of absorbance by the peptide bond, wasmonitored. From the peak area at this wavelength range a value for theabsorbance area per unit mass of peptide. Because all poly(Gln) peptideshave absorption characteristics at this wavelength that will bedominated by the poly(Gln) sequence, this “weight extinctioncoefficient” was used to calculate the weight concentrations ofsolutions of other poly(Gln) peptides of different length. Thebiotinyl-peptide was diluted into extension buffer to a finalconcentration of 10 nM, aliquoted, snap-frozen and stored at −80° C.

EXAMPLE 11 Extension Assay

For each replicate of each time point, the extension/storage buffer wasremoved from an aggregate-coated well and replaced by 100 μl of 10 nMbiotinyl-K₂Q₂₈K₂ peptide, after which the plate was incubated at 37° C.Kinetics data was collected by establishing individual time points inreverse temporal order. Thus, biotinyl-poly(Gln) peptide was introducedinto three wells (for reactions analyzed in triplicate) and the plateincubated at 37° C. sealed with an adhesive overlay. These wells providethe longest time-point data. At this time, the originalextension/storage buffer is retained for all other aggregate-coatedwells. At the next appropriate time, the plate was removed from the ovenand extension/storage buffer removed and discarded from the next set ofwells, and replaced by fresh 100 μl aliquots of 10 nM biotinyl-K₂Q₂₈K₂peptide. The plate was resealed and returned to 37° C. This process wasrepeated until all time points were added. After a final incubation toprovide the reaction time for the last-added (therefore earliest) timepoint, the entire plate was carried through the process described belowto develop and measure the signal.

Extension reactions were stopped simultaneously with 3 washes withextension buffer. After the 96-well plate was washed, it was incubated 1hr at room temperature with 100 μl per well of a 100 ng/mleuropium-streptavidin solution (EG&G Wallac, Gaithersburg, Md.) inextension buffer containing 0.5% BSA. The plate was then washed 3 timeswith extension buffer and the europium was released from streptavidin byaddition of 100 μl of enhancement solution (EG&G Wallac), as describedin Hemmila, et al., Europium as a Label in Time-ResolvedImmunofluorometric Assays, Anal. Biochem., vol. 137:335-343 (1984).After 5 min, europium was measured by a time-resolved fluorometry asdescribed in Diamandis, Immunoassays with Time-Resolved FluorescenceSpectroscopy: Principles and Applications, Clin. Biochem., 21:139-150(1988) in a Victor 2 (EG&G Wallac) microtiter plate reader using theprogrammed parameters for counting europium. Europium counts wereconverted to Fmoles europium using a standard curve obtained with aeuropium solution obtained from EG&G Wallac. Fmoles of europium wasconverted to Fmoles of biotinylated peptide using the manufacturer'sdetermination of 7 Eu³⁺ ions per streptavidin molecule.

The linearity of the assay of the invention is graphically shown inFIGS. 3 and 4. FIG. 3 is a line graph showing the dependence of the rateof the fast phase on biotinyl-poly(Gln) concentration. 20 ng/well ofimmobilized K₂Q₂₈K₂ aggregates were incubated with biotinyl-K₂Q₂₈K₂ inthe range from 1 to 30 nM. FIG. 4 is a pair of line graphs showing thecorrelation of the signal strength detected with the amount of poly(Gln)aggregate deposited in the microplate. In FIG. 4A, various amounts ofK₂Q₂₈K₂ aggregates were deposited in the microplate in the 25 pg-5 ngrange, and incubated 4 hrs with 10 nM of the biotinyl-K₂Q₂₈K₂ variousamounts of poly(Gln) aggregate (K₂Q₂₈K₂). In FIG. 4B, Microplate wellswere filled with 100 μl of 780 pg to 200 ng/ml K₂Q₄₇K₂ aggregates mixedwith 10 mg/ml lipid-extracted brain tissues (∘) or with extension buffer(▪) and incubated 6 hrs with 100 nM of the biotinyl-K₂Q₂₈K₂.Lipid-extracted brain tissue was obtained after sonication and fourtreatments with chloroform:methanol of small amount of brain tissue(≦0.5 g) as described (Current Protocols in Molecular Biology, Wiley).The error bars represent the standard deviation of 3 replicates.

To verify that the assay results would not be compromised due topossible biotinyl-peptide dissociation from the aggregates during theeuropium-streptavidin incubation, a test was run and showed thatbiotinyl-peptide deposited onto a poly(Gln) aggregate coated well doesnot dissociate appreciably even after 5 hr at 37° C.

EXAMPLE 12 Assay of the Invention with Monomeric poly(Gln) Peptides ofVarying Lengths

Solutions (10 nM) of disaggregated biotinyl-poly(Gln) peptides ofvarying lengths from Q₅ to Q₄₉ were incubated at 37° C. in wellscontaining immobilized Q₃₇ poly(Gln) peptide aggregates. Thesupernatants were discarded and the wells were washed and incubated atroom temperature with a solution of the europium complex ofstreptavidin. The wells were washed and the deposited europium releasedinto solution with a complexation buffer, then counted by time-resolvedfluorescence in a Victor 2 microplate fluorimeter.

The results, shown in FIG. 6, show that biotinylated poly(Gln) peptidesof different length were readily deposited onto immobilized poly(Gln)aggregates. The results further show that both the amplitude and therate of the initial, rapid phase of the extension reaction increase withincreasing poly(Gln) repeat length.

Peptides with repeat lengths of Q₅ and Q₁₀ exhibited only a small degreeof binding/extension. In contrast, peptides with repeat lengths of Q₁₅and Q₂₀ readily added to the pre-existing aggregates. As the poly(Gln)repeat length further increased up to Q₃₉, the ability to be recruitedinto the existing aggregate also increased. Further length increases,represented by the Q₄₄ and Q₄₉ peptides, did not add measurably to therate of extension.

Thus, the assay of the invention established that, while poly(Gln)repeat length contributes to extension activity, the repeat lengthconstraints are less severe for aggregate extension than for spontaneousnucleation. See Example 4 above and FIG. 8. In particular, the datashows that peptide elements as short as Q₁₅ are sufficient to supportthe recruitment of a poly(Gln) containing protein by a pre-existingaggregate.

EXAMPLE 13 Assay of the Invention with Aggregates of Differing Structure

The assay of example 11 was repeated except that six different versionsof Q₃₇ aggregates were immobilized and their capability to support therecruitment of additional monomeric poly(Gln) peptide was tested with abiotinyl-Q₂₈ peptide (K₂Q₂₈K₂).

FIG. 7 shows that different aggregated states of the same poly(Gln)sequence exhibit dramatically different abilities to serve as templatesfor heterologous extension by other poly(Gln) peptides. Six differentversions of two series of aggregates of the Q₃₇ peptide were prepared.One series of aggregates was prepared by incubating peptide at 37° C. togenerate the large, ordered aggregates of the prior art shown in FIG. 1.The other series was prepared as described in Example 6 above to produceaggregates of the invention as shown in FIG. 2. Sub-populations of theaggregates of both series were further processed by sonication with orwithout a following filtration step, as described in Example 7 above.For each of the resultant six aggregate preparations, the weightconcentration of aggregates was determined and equal weights ofaggregates were fixed to the wells of microtiter plates.

FIG. 7 shows that these aggregates, all derived from the same Q₃₇peptide, very considerably in their abilities to support heterologousextension by a poly(Gln) peptide. The data shown in FIG. 7 establishesthat aggregates of the invention, that is, formed with a freezing step,are more efficient in supporting extension than are the aggregatesformed without the freezing step. Additionally, the data shows that,within each of the two series, smaller particles are more efficient atsupporting extension than are larger ones, as sonicated plus filteredaggregates were more efficient than those that were sonicated withoutfiltering, which in turn were more efficient than non-sonicatedaggregates.

EXAMPLE 14 Assaying for Inhibitors of poly(Gln) Aggregation EXAMPLE 14AScreening Assay

Compound libraries were obtained with compounds dissolved in DMSO andarranged in deep-well plates in 96-well format. From these primarycompound library collections, “working stock” plates were prepared inwhich each well contained a compound at a concentration 10 times higherthan the desired, final screening assay concentration, in a solvent of50% DMSO and 50% PBS. In the example, compound concentrations in the“working stock” plate were about 1 mM, so that final assayconcentrations were about 100 μM. However, other concentrations can beused.

Assay plates with wells containing fixed polyGln aggregates wereprepared as described in Example 9. Wells were filled with 80 μl ofextension buffer and 10 μl of the working stock plate-compounds andincubated for 5 minutes. Control wells were also set up containing 80 μlof extension buffer and 10 μl of the 50/50 DMSO/PBS solvent. Additionalcontrol wells contained no immobilized aggregates. In the example, eachmicroplate working stock of compounds was tested on three platesdeveloped in parallel, in order to assess the reproducibility of theassay and gain greater confidence in the results. However, single plateor other numbers of replicate plates can alternatively be conducted.After the 5 minute incubation, 10 μl of 100 nM biotinyl-peptide storedin 5% DMSO was added to each well and the plate incubated for 45 mins at37° C. Extension reactions were stopped with three washes with extensionbuffer. The signal was then developed as described for the basicextension assay in Example 11.

Data was processed as follows. The Fmoles of biotinyl-polyGln bound inthe control wells lacking aggregate was subtracted from the signal forall other wells within the plate. The corrected signals for wellscontaining aggregate but no inhibitor were averaged. The signal for eachtest inhibitor was divided by this average and multiplied times 100 togenerate the percent amount of peptide incorporation compared tocontrol. This value was subtracted from 100 to generate the %inhibition. Finally, for each compound, the % inhibition values from thethree replicate plates were averaged and recorded. In the example,compounds delivering 50% inhibition or greater were considered hits.However, in principle any degree of inhibition significantly higher thanthe apparent inhibition of the bulk of the test compounds can beassigned as a hit. In practice, the value defining a “hit” is sometimesarbitrarily set in order to obtain a desired “hit rate”.

EXAMPLE 14B Determination of the IC50 Values of the IdentifiedInhibitors

As described above, 9.15 pmoles/well of synthetic aggregates wereimmobilized onto each well. After three washes in extension buffer,wells were incubated 5 minutes with various concentrations of a positivecompound, the higher concentration starting at 100 μM. Following the 5minute incubation, 10 μl of 100 nM of biotinyl-polyGIn peptides wereadded for 45 minutes. At the end of the incubation, the plate was washedthree times and the rest of the protocol was applied as described above.For each inhibitor, experiments were done in triplicate and the averagetaken to determine the IC50 value, the concentration of inhibitorrequired to give 50% inhibition of extension, compared to a controllacking any added compounds.

EXAMPLE 15 Aggregates Associated with Natural Disease

To confirm that the extension reaction observed for chemicallysynthesized poly(Gln) peptides as described in Example 10 is guided bythe same interactions as those operating in the aggregation ofdisease-related poly(Gln) proteins, aggregates of ataxin-3 (AT3), theprotein responsible for Machado-Josef Disease were prepared and tested.Chemically synthesized poly(Gln) peptides shorter than the 35-40 repeatlength pathological cutoff are capable of forming ordered aggregates invitro. See FIG. 8. Similarly, AT3 with a normal length poly(Gln)sequence is known to be capable of aggregating in vitro.

AT3(Q₂₇) from E. coli extracts was purified and allowed to aggregate. Ahuman recombinant expression construct for the GST fusion with ataxin-3(Q₂₇) [AT3(Q₂₇)], obtained from Dr. H. Paulson (U. of Iowa), contains anIPTG inducible tac promoter and a thrombin cleavage site providing forthe efficient removal of the amino terminal GST fusion from the AT3(Q₂₇)protein. The construct was transformed into the E. coli expressionstrain BL21. The E. coli was grown at 37° C. to late log phase (O.D.600=0.8−0.9) before a 1 mM IPTG induction. Upon induction, the growthtemperature was reduced to 32° C. to minimize the deposition of theAT3(Q₂₇) into insoluble inclusion bodies. The culture was induced for 2hours before the cells were harvested by centrifugation. The pelletedcells were then resuspended in sonication lysis buffer (50 mM Tris, 50mM NaCl, and 5 mM EDTA, pH 8) supplemented freshly with 0.15 mM PMSF,1.46 μM pepstatin A and 2.4 μM leupeptin (Sigma). The cells weresubsequently lysed by sonication and the insoluble material removed bycentrifugation.

Purification of the AT3(Q₂₇) protein was achieved through glutathionesepharose spin filtration chromatography (Amersham Pharmacia,Piscataway, N.J.) followed by on-resin cleavage with thrombin (Novagen,Madison, Wis.) to liberate the ataxin-3 from the bound GST fusionprotein. As the final purification step, the resultant digestsupernatant containing the cleaved ataxin-3 and thrombin proteins wasapplied to a S-300 sephacryl (Amersham Pharmacia) gel filtration column.The cleaved AT3(Q₂₇) eluted as a single peak at the void volume of theS-300 column, consistent with an average globular protein molecularweight of greater than or equal to 1,500 kDa. This indicates that theataxin protein at this stage of the purification is a water solubleaggregate containing at least 30 molecules of AT3.

SDS-PAGE and western blot screenings of the void volume peak revealed asingle band at ˜42 kDa that was immunoreactive with both an ataxin-3polyclonal antibody and the polyglutamine specific 1C2 monoclonalantibody (Chemicon, Temecula, Calif.). These fractions were pooled andincubated at room temperature for six days to complete the aggregationprocess. A sample of the aggregated AT3(Q₂₇) run on SDS-PAGE revealed atrio of SDS resistant, Coomassie brilliant blue staining bands that wereunable to be transferred and screened by western blot. Subsequent dotblot screening verified that the aggregates retain immunoreactivity withthe 1C2 antibody. The aggregates were pelleted by centrifugation andresuspended in 1× PBS pH 7.4 buffer and stored at 4° C. Microtiterplates coated with aggregated AT3(Q₂₇) were prepared in analogy to theabove protocol described in Example 8 for chemically synthesizedpoly(Gln) peptides.

The ability of aggregates of AT3(Q₂₇) to be extended by biotinyl-K₂Q₂₈K₂when the aggregates are fixed to microplate wells was studied. In thisstudy, the amount of AT3 aggregates fixed to the wells was adjusted sothat the amount of poly(Gln) on the wells was the same as the amount ofaggregated K₂Q₂₈K₂ on the same plate. FIG. 5 shows that the kinetics ofextension of both aggregates by biotinyl-K₂Q₂₈K₂ proceed with similarparameters. This suggests that the in vitro extension process that isobserved for the synthetic poly(Gln) aggregates described above isrelevant to the natural disease processes of expanded CAG repeatdiseases, and that tissue-derived aggregates of disease-associatedproteins are observable using the assay of the invention.

Each of the patents and scientific articles cited herein is incorporatedherein by reference. Although the above description contains manyspecificities, they should not be interpreted as limitations on thescope of the invention, but rather as illustrations. One skilled in theart will understand that many variations of the invention are possibleand that these variations are to be included within the scope of thefollowing claims.

1. A method for making an aggregate of aggregation-prone polypeptidescomprising obtaining a solution of the aggregation-prone polypeptides,freezing the solution containing the polypeptides, incubating the frozenpolypeptides in a frozen state, and permitting the aggregates to form.2. The method of claim 1 wherein the aggregation-prone polypeptidecontains a polyglutamine repeat sequence.
 3. The method of claim 2wherein the solution comprises polypeptides comprising a polyglutaminerepeat sequence of at least Q₃₅.
 4. The method of claim 1 wherein thefreezing is a snap freezing.
 5. The method of claim 1 which furthercomprises sonicating the aggregates.
 6. The method of claim 5 whichcomprises, after the sonicating, filtering the aggregates.
 7. The methodof claim 6 wherein the filtration is through a membrane filter.
 8. Themethod of claim 7 wherein the membrane filter is a 1.2 μm membranefilter.
 9. An in vitro produced aggregate that is made by the method ofclaim
 1. 10. An in vitro produced aggregate that is made by the methodof claim
 5. 11. An in vitro produced aggregate that is made by themethod of claim
 6. 12. A method for dissolving or disaggregating apolypeptide comprising combining the polypeptide in a mixture oftrifluoroacetic acid (TFA) and hexafluoroisopropanol (HFIP) andpermitting the polypeptide to dissolve or disaggregate in the mixture.13. The method of claim 12 wherein the polypeptide is a polyglutaminerepeat polypeptide.
 14. The method of claim 13 wherein the polyglutaminerepeat polypeptide has a glutamine repeat sequence of Q₃₅ or more. 15.The method of claim 12 which further comprises removing the TFA and HFIPand resolubilizing the polypeptide in water.
 16. The method of claim 12wherein the ratio of TFA and HFIP in the mixture is between 20:1 and1:20.
 17. The method of claim 16 wherein the ratio is between 5:1 and1:5.
 18. The method of claim 17 wherein the ratio is about 1:1.